title: Construction and characterization of normalized cDNA libraries by 454 pyrosequencing and estimation of DNA methylation levels in three distantly related termite species.
abstract: In termites, division of labor among castes, categories of individuals that perform specialized tasks, increases colony-level productivity and is the key to their ecological success. Although molecular studies on caste polymorphism have been performed in termites, we are far from a comprehensive understanding of the molecular basis of this phenomenon. To facilitate future molecular studies, we aimed to construct expressed sequence tag (EST) libraries covering wide ranges of gene repertoires in three representative termite species, Hodotermopsis sjostedti, Reticulitermes speratus and Nasutitermes takasagoensis. We generated normalized cDNA libraries from whole bodies, except for guts containing microbes, of almost all castes, sexes and developmental stages and sequenced them with the 454 GS FLX titanium system. We obtained >1.2 million quality-filtered reads yielding >400 million bases for each of the three species. Isotigs, which are analogous to individual transcripts, and singletons were produced by assembling the reads and annotated using public databases. Genes related to juvenile hormone, which plays crucial roles in caste differentiation of termites, were identified from the EST libraries by BLAST search. To explore the potential for DNA methylation, which plays an important role in caste differentiation of honeybees, tBLASTn searches for DNA methyltransferases (dnmt1, dnmt2 and dnmt3) and methyl-CpG binding domain (mbd) were performed against the EST libraries. All four of these genes were found in the H. sjostedti library, while all except dnmt3 were found in R. speratus and N. takasagoensis. The ratio of the observed to the expected CpG content (CpG O/E), which is a proxy for DNA methylation level, was calculated for the coding sequences predicted from the isotigs and singletons. In all of the three species, the majority of coding sequences showed depletion of CpG O/E (less than 1), and the distributions of CpG O/E were bimodal, suggesting the presence of DNA methylation.
authors.affiliation: Graduate School of Environmental Science, Hokkaido University, Sapporo, Japan ; School of Biological Sciences, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Shuji
authors.initials: S
authors.lastname: Shigenobu
authors.forename: Dai
authors.initials: D
authors.lastname: Watanabe
authors.forename: Kouhei
authors.initials: K
authors.lastname: Toga
authors.forename: Ryota
authors.initials: R
authors.lastname: Saiki
authors.forename: Keisuke
authors.initials: K
authors.lastname: Shimada
authors.forename: Thomas
authors.initials: T
authors.lastname: Bourguignon
authors.forename: Nathan
authors.initials: N
authors.lastname: Lo
authors.forename: Masaru
authors.initials: M
authors.lastname: Hojo
authors.forename: Kiyoto
authors.initials: K
authors.lastname: Maekawa
authors.forename: Toru
authors.initials: T
authors.lastname: Miura
mesh_terms: D000818:Animals; D019295:Computational Biology; D019175:DNA Methylation / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D020224:Expressed Sequence Tags; D015723:Gene Library; D014644:Genetic Variation; D005828:Genetics, Population; D006608:Hierarchy, Social; D020049:Isoptera / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D013045:Species Specificity
chemical_list: D017931:DNA Primers